Fungal infections remain among the most difficult to treat. Innate recognition of microbes is critical for effective host responses, and is often accomplished by myeloid cells including macrophages. Bacterial PAMPs are recognized by cell surface, endosomal, and cytoplasmic receptors. Cytoplasmic recognition of bacterial PAMPS has been suggested to enable the innate immune system to distinguish pathogens from noninvasive commensals. The fungal PAMPs ??glucan and mannan are recognized by cell surface C-type lectin receptors, but no cytoplasmic receptors have been identified for fungal-specific PAMPs. Early studies of intracellular detection of bacterial cell wall components utilized direct cytoplasmic introduction of PAMPs such as lipopolysaccharide (LPS) to infer the existence of cytoplasmic receptors. Depending on the PAMP, cytoplasmic introduction of such components can induce a Nuclear Factor kappa B (NF-kB) response (via the NOD1 or NOD2 receptors) and/or the activation of inflammasomes. We employed this cytoplasmic introduction approach using material extracted from the fungal cell wall preparation zymosan (?soluble zymosan?) and found that it can potently trigger an NF-kB response. Further, we have found that this response?which requires the cytoplasmic introduction of soluble zymosan?is genetically independent of the Toll-like receptor (TLR) signaling adaptor MyD88 and the C-type lectin (CLR) signaling components Syk and CARD9. Together, these data argue that known cell surface sensing mechanisms do not mediate this effect. Partial purification of the activity has identified a protease-sensitive soluble fraction that displays dose-dependent NF-kB responses without activating inflammasome responses (IL-1? secretion). As LPS does not have these properties upon transfection (LPS activates both NF-kB and IL-1? responses and is not protease-sensitive), these and other experiments strongly argue against the possibility that LPS contamination of zymosan is the source of this activity. For brevity, we refer to this activity as the CSZR (cytoplasmic soluble zymosan response). Ultimate proof of its existence and importance requires the molecular identification of the relevant receptor and microbial inducer. In this high-risk, exploratory R21 proposal, we will use classical biochemical purification and mass spectrometry to purify and identify the inducing ligand and we will perform a whole-genome CRISPR screen in immortalized bone marrow- derived macrophages to identify candidates for the cognate ligand receptor. Significance/potential impact: This work has the potential to reveal a previously unrecognized intracellular sensing mechanism for a fungal PAMP that has the potential to transform our understanding of innate immunity to fungi. Such knowledge may yield new approaches for the effective treatment of invasive fungal disease, an increasing global threat to human health.